1. Field of the Invention
This specification relates to an apparatus for forming an alignment layer, and particularly, to an aqueous-dispersed fluorescent nanoprobe, which contains benzene compound exhibiting fluorescence, sulfonated benzene compound exhibiting fluorescence by reaction with hydrogen peroxide and hydrophobic organic base deriving the fluorescence reaction so as to be applicable for real-time detection of hydrogen peroxide, and a fabrication method thereof.
2. Background of the Invention
Reactive oxygen species (ROS) are essentially generated within a body of aerobe. However, if it is excessively generated or accumulated within the body, it is closely connected with a mechanism, which causes extensive inflammatory diseases, such as cancers, diabetes, Alzheimer's disease, arteriosclerosis, arthritis and the like. Hence, in vivo and in vitro detections of the ROS are very important.
Examples of the ROS include singlet oxygen (1O2), hydroxyl radical (OH−), superoxide anion (O2−) and hydrogen peroxide (H2O2). The others except for the hydrogen peroxide is extremely unstable in a physiological environment, thus to be easily reduced into hydrogen peroxide or water. Hence, in diagnosis of diseases involved in overexpression of ROS, the most advantageous method is to detect hydrogen peroxide, which exhibits the highest stability among ROS and exists in a body in the highest concentration (1×10−7 M), in vivo and in vitro.
As representative methods for detecting hydrogen peroxide, a method using fluorescence [Document 1: Novuaki Soh, Anal. Bioanal. Chem. 386: 532-543 (2006)] and a method using chemiluminescence [Document 2: Ahao Lu et al., Trends Anal. Chem. 25: 985-995 (2006), Document 3: Zhenyu Zhang et al. Anal. Chim. Acta 541: 37-47 (2005)] are broadly used.
The detection of the hydrogen peroxide using the chemiluminescence does not require for an irradiation of excitation light, so any background is fluorescence or interference due to the excitation light is not caused. Accordingly, such method has advantage of acquiring high signal-to-noise ratio in spite of its low luminescence efficiency as compared to the fluorescence detection method. Hence, it is very favorable for the in vivo detection. However, such method needs a high-sensitivity detector due to a low absolute signal intensity and is hard to select (determine) a detection time due to disappearance of the luminescence within a short time. Consequently, the detection method using the chemiluminescence has limitation in fabrication of a low-cost sensor for in vivo and in vitro detections.
The fluorescence detection needs excitation light. As compared with the chemiluminescence detection, the fluorescence detection is disadvantageous in that the background fluorescence interferes with detection of a signal. However, the high-sensitivity detector is not needed by virtue of a strong signal and an accumulated signal unlike the chemiluminescence, so the fluorescence detection has no difficulty in determining detection conditions, thereby being effective to fabricate various sensors. The fluorescence detection for hydrogen peroxide is executed by employing a method for measuring fluorescence changes in response to oxidation of fluorescent molecules, which are easily oxidized by the hydrogen peroxide [Document 4: Naoki Umezawa et al. Angew. Chem. Int. Ed. 38: 2899-2901 (1999)], or a method for coupling substituent, which is easily separated by hydrogen peroxide, to fluorescent molecules and measuring changes in fluorescence properties caused by the separation of the substituent [Document 5: Evan W Miller et al., Nat. Chem. Biol. 3: 263-267 (2007), Document 6: Hatsuo Maeda et al., Angew. Chem. 116: 2443-2445 (2004)]. The fluorescence detection method has many advantages, but is not suitable for real-time detection due to taking a long reaction time over 1 hour. Hence, it is the urgent problem to reduce the reaction time for real-time detection.